Construction of an Exonuclease III-Propelled Integrated DNAzyme Amplifier for Highly Efficient microRNA Detection and Intracellular Imaging with Ultralow Background.

DNAzyme amplifiers show great potential in bioanalysis but their operation in living cells still remains a challenge because of the intrinsic low-abundance analytes and the undesired background interference. Herein, we constructed a simple yet versatile exonuclease III (Exo-III)-powered cascade DNAzyme amplifier with an ultralow background for highly sensitive and selective microRNA assay in vitro and even in living cells. The present DNAzyme amplifier relies on only one DNAzyme-functionalized hairpin (HP-Dz) probe that is grafted with two exposed subunits of an analyte recognition strand, through which false enzymatic digestion and DNAzyme leakage could be substantially expelled. These protruding ssDNA strands could cooperatively recognize and efficiently bind with the miR-21 analyte, releasing the blunt 3'-terminus for Exo-III digestion and then regenerating miR-21 for a new round of HP-Dz activation. This leads to the production of numerous DNAzyme units for catalyzing the cleavage of the fluorophore/quencher-tethered substrate and yielding an enormously amplified fluorescence readout. The successive Exo-III-mediated analyte regeneration and DNAzyme-involved signal amplification facilitate their ultrasensitive miR-21 assay in vitro and intracellular miR-21 imaging. Note that the present DNAzyme module could be facilely substituted with another versatile HRP-mimicking DNAzyme, thus enabling the colorimetric assay of miR-21 with naked eye observation. Overall, this robust Exo-III-propelled cascaded DNAzyme amplifier provides more general and versatile approaches for understanding miRNA functions of related biological events.